Positive-displacement pipette

A DNA extraction protocol that has proved useful for most ancient tissues is a modification of the protocol initially published by Blin and Stafford.20 Approximately 0.1 g of small pieces of soft tissue is added to 5 ml of extraction buffer containing 10 mM Tris-HCl (pH 8.0), 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM NaCl, 1% (w/v) sodium dodecyl sulfate (SDS), 10 mg/ml dithiothreitol (DTT), and 0.5 mg/ml proteinase K. Incubation at 37° with gentle agitation overnight will allow most or all of the tissue to go into solution. An equal volume of phenol, equilibrated with 1 M Tris-HCl (pH 8.0), is added. When the phenol is being equilibrated, care should be taken to use uncontaminated Tris buffer and to measure the pH only on aliquots that are removed from the water phase and then discarded. Two phenol extractions and one chloroform extraction are performed, and the water phase is concentrated and purified on a Centricon 30 microconcentrator (Amicon, Danvers, MA). The reten-tate can be stored frozen, preferably in a few aliquots. In all cases solutions should be manipulated with DNA-free positive displacement pipettes. 

Obtain double-stranded amplifications of all samples with the same pair of primers used for perpendicular DGGE (one should include a GC clamp). Use total reaction volumes of 25 or 12.5 pi. Run on agarose minigels and take plugs by puncturing with clean, positive displacement pipettes. Save the plugs in 250 pi of LTE buffer. 

The simplest and most cost-effective injection system is the Drummond Nanoject (Laser Laboratory Systems). This is essentially a motor-driven positive displacement pipette and is available in versions that deliver fixed (46 nl) or variable (4.6-73.6 nl) volumes. The setting of the fixed model can be changed by substituting a chip in the controller, but the flexibility of the variable version is a distinct advantage. The injector can be operated by hand via a control box, but the optional foot switch is worthwhile if more than a small number of oocytes are to be injected in a session. 

Delivery of an aliquot through a pipette by positive displacement using a syringe or a micro pump that delivers precisely defined volume increments. 

Positive-liquid-displacement pipettes are used for specimen handling in most discrete automated systems. With them, specimens, calibrators, and controls are delivered by a single pipette to the next stage in the analytical process. 

While fractions or samples can be removed from the centrifuge tubes using a pipette it is possible with appropriate equipment to pierce the base of a plastic tube to allow the contents to drip slowly out. Alternatively the contents of the tube may be displaced by slowly injecting a dense solution to the bottom of the tube (Figure 3.44) or by puncturing the tube in the appropriate position and withdrawing the desired band with a syringe. 

The pipettes are all based on air displacement with a simple plunger and are provided with non-wettable plastic (usually polypropylene) disposable tips to contain the solution, preventing any contamination of the pipette itself. Most micropipettes have a double action plunger system, i.e. calibration and overshoot positions, which ensures that the sample is completely dispensed. There are many manufacturers offering a complete range of volumes in addition some have available a selection of pipettes with adjustable volumes. 

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